Curcumin inhibits the mammalian target of rapamycin-mediated signaling pathways in cancer cells

Abstract

Curcumin (diferuloylmethane), a polyphenol natural product of the plant Curcuma longa, is undergoing early clinical trials as a novel anticancer agent. However, the anticancer mechanism of curcumin remains to be elucidated. Here we show that curcumin inhibited growth of rhabdomyosarcoma cells (Rh1 and Rh30) (IC₅₀ = 2–5 μM) and arrested cells in G₁ phase of the cell cycle. Curcumin also induced apoptosis and inhibited the basal or type I insulin-like growth factor-induced motility of the cells. At physiological concentrations (˜2.5 μM), curcumin rapidly inhibited phosphorylation of the mammalian target of rapamycin (mTOR) and its downstream effector molecules, p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), in a panel of cell lines (Rh1, Rh30, DU145, MCF-7 and Hela). Curcumin also inhibited phosphorylation of Akt in the cells, but only at high concentrations (>40 μM). The data suggest that curcumin may execute its anticancer activity primarily by blocking mTOR-mediated signaling pathways in the tumor cells. © 2006 Wiley-Liss, Inc.

Curcumin (diferuloylmethane) is a polyphenol natural product isolated from turmeric, a powder produced from the rhizome of the plant Curcuma longa.1 Curcumin has been shown to produce a wide range of medicinal benefits in the human body and has proven to be a powerful therapeutic agent against many human disease processes.1 In recent years, curcumin has garnered interest as a potential anticancer agent. A number of studies have shown that curcumin exerts strong anticancer effects against a broad range of human cancer cells, including those derived from breast cancer,2, 3, 4 prostate cancer,4, 5, 6 colon cancer,7, 8 hepatocellular carcinoma,9 T-cell leukemia,10 B-cell lymphoma,11 mantle cell lymphoma,12 basal cell carcinoma,13 melanoma,14 renal carcinoma,15 and neuroblastoma.16

Curcumin can affect and disrupt virtually every major stage of carcinogenesis, including cell proliferation, growth, survival, angiogenesis and metastasis.1 The molecular mechanisms of the action that curcumin employs to affect these processes are numerous and varied, depending on the cancer cell type.1 These mechanisms include inhibition of ornithine decarboxylase (ODC),2 downregulation of c-myc9, 11 and cyclin D1,4, 8 inhibition of the signaling pathways of nuclear factor kappa B (NF-κB),2, 6, 11, 12, 17 activating protein-1,11, 12 protein tyrosine kinases,3, 18 Akt,7, 15 and protein kinase C (PKC).19 While it is very possible that curcumin may target each of these individual molecules in a cell-type and cell-environment dependent manner, it appears to be more logical to conclude that curcumin directly affects a few major targets and this indirectly impacts all of the other factors.

The mammalian target of rapamycin (mTOR), a serine/threonine kinase, lies downstream of the type I insulin-like growth factor receptor-phosphatidylinositol 3′ kinase-Akt, and positively regulates phosphorylation of ribosomal p70 S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1).20 Inhibition of mTOR by rapamycin results in hypophosphorylation of 4E-BP1. Subsequently, hypophosphorylated 4E-BP1 tightly binds to eIF4E, and prevents association of eIF4E with eIF4G and formation of the eIF4F initiation complex, thereby inhibiting cap-dependent translation of mRNA.21, 22 In addition, inhibition of mTOR by rapamycin inactivates S6K1, blocking translation of mRNA species containing 5′ terminal oligopyrimidine tracts,23, 24 though this remains controversial.25, 26 mTOR is also involved in the regulation of cyclins D1/A,27, 28, 29 cyclin-dependent kinases,30, 31 cyclin-dependent kinase inhibitors (p21^Cip1 and p27^Kip1),32, 33, 34 ODC,35, 36 c-myc,37 retinoblastoma protein,38 RNA polymerase I/II/III-transcription and translation of rRNA and tRNA,39, 40 protein phosphatases (PP2A, PP4 and PP6),41, 42 hypoxia-inducible factor-1α,43 vascular endothelial growth factor,44 CLIP-170,45 Akt,46 PKC47 and NF-κB.48 Therefore, mTOR has been implicated as a central regulator of cell proliferation, growth, angiogenesis, survival and motility. Of particular interest is that among the proteins regulated by mTOR, a number of them, such as cyclin D1, ODC, c-myc, NF-κB, Akt and PKC, are also targeted by curcumin. This prompted us to study whether curcumin inhibits mTOR signaling.

Here we show that curcumin inhibits cell growth, induces apoptosis and suppresses motility of rhabdomyosarcoma cells. Furthermore, at physiologically relevant concentrations (˜2.5 μM), curcumin inhibits the mTOR-mediated phosphoryaltion of S6K1 and 4E-BP1 in a panel of cell lines, including Rh1, Rh30, DL1145, MCF-7 and Hela cells. Only at higher concentrations (>40 μM) does curcumin inhibit phosphorylation of Akt. The findings suggest that curcumin may execute its anticancer activity by primarily targeting mTOR-mediated signaling pathways. Curcumin may represent a new class of mTOR inhibitor.

DMEM, Dulbecco's Modified Eagle Medium; 4E-BP1, eukaryotic initiation factor 4E (eIF4E) binding protein 1; FBS, fetal bovine serum; IGF-I, type I insulin-like growth factor; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor kappa B; ODC, ornithine decarboxylase; PBS, phosphate-buffered saline; PKC, protein kinase C; S6K1, p70 S6 kinase 1.

Material and methods

Materials

Curcumin (Sigma, St. Louis, MO) was dissolved in 100% ethanol to prepare a stock solution (10 mM), aliquoted and stored at −20°C. Type I insulin-like growth factor (IGF-I) (PeproTech, Rocky Hill, NJ) was rehydrated in 0.1 M acetic acid to prepare a stock solution (10 μg/ml), aliquoted and stored at −80°C. RPMI 1640 and Dulbecco's Modified Eagle Medium (DMEM) were purchased from Mediatech (Herndon, VA). Fetal bovine serum (FBS) was from Hyclone (Logan, UT), whereas 0.05% Trypsin-EDTA was from Invitrogen (Grand Island, NY). Enhanced chemiluminescence solution was from PerkinElmer Life Science (Boston, MA). The following antibodies were used: rabbit anti-phospho-p70 S6K1 (Thr389), mouse anti-p70 S6K1, rabbit anti-phospho-Akt (Thr308), rabbit anti-phospho-Akt (Ser473), mouse anti-Akt (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-4E-BP1 (Zymed, South San Francisco, CA), rabbit anti-phospho-4E-BP1 (Thr37/46), rabbit anti-phospho-4E-BP1 (Ser65), rabbit anti-phospho-4E-BP1 (Thr70), rabbit anti-phospho-mTOR (Ser2448), rabbit anti-phospho-mTOR (Ser2481) (Cell Signaling, Beverly, MA), mouse anti-mTOR (26E3) antibody,49 mouse anti-β-tubulin (Sigma), goat anti-mouse IgG-horseradish peroxidase and goat anti-rabbit IgG-horseradish peroxidase (Pierce, Rockland, IL).

Cell lines and cultures

Cell lines from human rhabdomyosarcoma (Rh1 and Rh30) (St. Jude Children's Research Hospital, Memphis, TN) were grown in antibiotic-free RPMI 1640 medium supplemented with 10% FBS at 37°C and 5% CO₂. Both express mutant p53 alleles (Rh1 Tyr²²⁰ → Cys, Rh30 Arg²⁷³ → Cys).50 Human breast carcinoma (MCF-7), cervical adenocarcinoma (Hela) and prostate carcinoma (DU145) cells (American Type Culture Collection, Manassas, VA) were grown in antibiotic-free DMEM supplemented with 10% FBS at 37°C and 5% CO₂. For experiments where cells were deprived of serum, cell monolayers were washed with phosphate-buffered saline (PBS) and incubated in the serum-free DMEM.

Cell proliferation assay

Rh1 and Rh30 cells were seeded in RPMI 1640 supplemented with 10% FBS in 6-well plates at a density of 5 × 10⁴ cells/well (in triplicate) and the cells were grown overnight at 37°C in a humidified incubator with 5% CO₂. Curcumin (0–40 μM) was added the next day. After incubation for 6 days, images were taken with an Olympus inverted phase-contrast microscope equipped with the Quick Imaging system. Cells were then trypsinized and enumerated using a Brightline hemacytometer.

Cell cycle analysis

Rh1 and Rh30 cells were seeded in 100 mm dishes at a density of 1 × 10⁶ cells/dish in RPMI 1640 supplemented with 10% FBS and were grown overnight at 37°C in a humidified incubator with 5% CO₂. Cells were then treated with 20 μM curcumin for 24 h. Cell cycle analysis was performed on the treated cells using the Cellular DNA Flow Cytometric Analysis Kit (Roche Diagnostics Corp., Indianapolis, IN). Cells treated with vehicle alone (100% ethanol) were used as a control.

Apoptosis assay

Rh1 and Rh30 cells were seeded in 100-mm dishes at a density of 1 × 10⁶ cells/dish in RPMI 1640 supplemented with 10% FBS and were grown overnight at 37°C in a humidified incubator with 5% CO₂. Cells were treated with 20 μM curcumin for 72 h, followed by apoptosis assay using the Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, San Diego, CA). Cells treated with vehicle alone (100% ethanol) were used as a control.

Cell motility assay

A monolayer of Rh1 or Rh30 cells were grown in 6-well plates to ˜80% confluence, and were serum-starved in DMEM for 24 h. Cell motility was assessed by the wound healing assay.51 Migration was initiated by removing a portion of the cell layer by scratching with a single-edge razor blade cut to ˜27 mm in length. The scratch began at the diameter of the dish and extended over an area ˜10 mm wide. The medium was changed to remove floating or damaged cells. Cells were pretreated with or without curcumin (20 μM) for 2 h, followed by stimulation with or without IGF-I (10 ng/ml) for 22 h. Cells migrated over the denuded area were observed and photographed with an Olympus inverted phase-contrast microscope equipped with the Quick Imaging system. The number of cells migrating per millimeter of scratch was counted.

Western blot analysis

Cells were seeded in 6-well plates in RPMI 1640 (for Rh1 and Rh30) or DMEM (for DU145, MCF-7 and Hela) supplemented with 1% FBS for 2 h. After attaching to the wells, the cells were then serum-starved with DMEM for 22 h at 37°C in a humidified incubator with 5% CO₂. Cells were then treated with 0–40 μM curcumin for 2 h, or with 20 μM curcumin for 0–24 h, followed by stimulation/nonstimulation with IGF-I (10 ng/ml). Subsequently, cells were briefly washed with cold PBS. On ice, cells were lysed in RIPA buffer [50 mM Tris, pH 7.2; 150 mM NaCl; 1% sodium deoxycholate; 0.1% SDS; 1% Triton-X 100; 10 mM NaF; 1 mM Na₃VO₄; protease inhibitor cocktail (1:1,000, Sigma, St. Louis, MO)]. Lysates were sonicated for 10 s and centrifuged at 14,000 rpm for 10 min at 4°C. Protein concentration was determined by bicinchoninic acid assay with bovine serum albumin as standard (Pierce, Rockford, IL). Equivalent amounts of protein were separated on 7.5–12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were incubated with PBS containing 0.05% Tween 20 and 5% nonfat dry milk to block nonspecific binding and were incubated with primary antibodies, then with appropriate secondary antibodies conjugated to horseradish peroxidase. We used primary antibodies to rabbit anti-phospho-p70 S6K1 (Thr389), mouse anti-p70 S6K1, rabbit anti-phospho-Akt (Thr308), rabbit anti-phospho-Akt (Ser473), mouse anti-Akt (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-4E-BP1 (Zymed, South San Francisco, CA), rabbit anti-phospho-4E-BP1 (Thr37/46), rabbit anti-phospho-4E-BP1 Ser65, rabbit anti-phospho-4E-BP1 (Thr70), rabbit anti-phospho-mTOR (Ser2448), rabbit anti-phospho-mTOR (Ser2481) (Cell Signaling, Beverly, MA) and mouse anti-mTOR (26E3) antibody.49 Immunoreactive bands were visualized by using Renaissance chemiluminescence reagent (Perkin-Elmer Life Science, Boston, MA). To check the amount of protein loaded, the immunoblots were treated with stripping solution (62.5 mM Tris buffer, pH 6.7, containing 2% SDS and 100 mM β-mercaptoethanol) for 30 min at 50°C and incubated with mouse monoclonal anti-β-tubulin antibody (Sigma) followed by horseradish peroxidase-coupled goat anti-mouse IgG (Pierce).

Statistical analysis

Data were expressed as means ± SD and statistically subjected to Student's unpaired t-test. A level of P < 0.05 was considered to be significant.

Results

Curcumin inhibits the proliferation of rhabdomyosarcoma cells and arrests the cells in the G₁/G₀ phase of the cell cycle

Due to the amazing success that curcumin displayed against many forms of human cancer, we decided to investigate its effects on rhabdomyosarcoma cells, a typical cell type with dysregulated IGF-I signaling.52, 53 To perform the experiments, we selected concentrations of curcumin that were comparable to those used in previously reported studies as well as concentrations that would be physiologically relevant. Phase I clinical trials have shown that high doses of curcumin can be delivered to patients with virtually no deleterious side-effects (8 g/day) (1.77 ± 1.87 μM average peak serum concentration.54 We therefore chose low micromolar concentrations of curcumin to investigate the effect of the compound on the proliferation/growth of rhabdomyosarcoma cells. Human rhabdomyosarcoma cell lines Rh1 and Rh30, which represent the two major forms of the cancer, embryonal and alveolar, respectively, were treated with curcumin (0–40 μM) for 6 days in RPMI 1640 medium supplemented with 10% FBS. As shown in Figure 1, curcumin not only exhibited a cytostatic effect at low micromolar concentrations, but also exerted a cytotoxic effect at the higher micromolar concentrations, as the original 50,000 seeded cells almost died out at 20 and 40 μM concentrations of curcumin after exposure for 6 days (Fig. 1a). The IC₅₀ of curcumin was ˜2.7 μM for Rh1 and ˜5.1 μM for Rh30, respectively (Fig. 1b).

Figure 1. Curcumin inhibits growth of rhabdomyosarcoma cells in a dose-dependent manner. Rh1 and Rh30 cells were seeded in 10% FBS-RPMI 1640 on 6-well plates (5 × 10⁴ cells/well) in triplicate. Curcumin (0–40 μM) was added to the medium the next day. After incubation for 6 days, (a) cells were photographed with an Olympus inverted phase-contrast microscope equipped with Quick Imaging system, Bar = 50 μm; or (b) cells were trypsinized and enumerated using a hemacytometer. Results are means ± SD and are pooled from three independent experiments. *P < 0.05 (unpaired Student's t-test).

Because cell proliferation and growth are controlled by the progression of the cell through the well-defined stages of the cell cycle, next we determined the effects of curcumin on the cell cycle progression of rhabdomyosarcoma cells through the use of propidium iodide staining and flow cytometry analysis. As shown in Figure 2, curcumin (20 μM, 24 h) effectively arrested both Rh1 and Rh30 cells in the G₁/G₀ phase of the cell cycle. For Rh1 cells, curcumin treatment significantly increased the proportion of cells in the G₁/G₀ phase from 48.4% to 66.2%, while for Rh30, the fraction of cells in the G₁/G₀ phase from 42.8% to 54.9%. This is an interesting finding because both Rh1 and Rh30 cells express mutant p53 alleles (Rh1 Tyr²²⁰→Cys, Rh30 Arg²⁷³→Cys), losing the function of p53.50 Therefore, it appears that curcumin is able to arrest cells in the G₁/G₀ phase and inhibit the proliferation of rhabdomyosarcoma cells in a p53-independent manner.

Figure 2. Curcumin arrests rhabdomyosarcoma cells in G₁ phase of the cell cycle. Rh1 and Rh30 cells were seeded in 100-mm dishes (1 × 10⁶ cells/dish) in 10% FBS-RPMI 1640 and grown overnight. Cells were treated with 20 μM curcumin for 24 h, followed by cell cycle analysis using the Cellular DNA Flow Cytometric Analysis Kit (Roche). Cells treated with vehicle (ethanol) were used as a control. Results are means ± SD and are pooled from three independent experiments. *P < 0.05 (unpaired Student's t-test).

Curcumin induces p53-independent apoptosis of rhabdomyosarcoma cells

Over 50% of human cancers, including rhabdomyosarcoma, contain a mutated form of p53, one of the most important tumor suppressor proteins present in cells.55 Wild-type p53 responds to DNA damage in cells and can arrest growth of the cells to allow time for DNA-repair to occur, or can induce apoptosis of the cells with irreparable DNA damage.55 Cancer cells, especially those with p53 mutations, are able to escape cell cycle arrest and apoptosis despite their large number of genetic mutations/malfunctions.55 Because of mutations of p53 in Rh1 and Rh30 cells, we examined whether curcumin can induce apoptosis of the rhabdomyosarcoma cells. Apoptosis was determined by the ApoAlert FACS analysis. We found that treatment with curcumin for 72 h induced apoptosis of Rh1 and Rh30 cells in a dose-dependent manner (data not shown), as seen in Figure 1a. A representative FACS assay result is shown in Figure 3a. Exposure to curcumin (20 μM, 72 h) induced significant increase of the proportion of cells positive for annexin V and propidium iodide (Rh1 - 38.8%), as compared to nontreated cells (Rh1 - 15.72%). Treatment with the compound increased the number of Rh1 cells undergoing apoptosis by ˜2.48-fold. Similar results were also seen in Rh30 cells (Fig. 3b). The results suggest that curcumin can induce p53-independent apoptosis of the rhabdomyosarcoma cells.

Figure 3. Curcumin induces apoptosis of rhabdomyosarcoma cells in a p53-independent manner. Rhabdomyosarcoma (Rh1, Tyr²²⁰ → Cys²²⁰; Rh30, Arg²⁷³ → Cys²⁷³) cells were seeded at a density of 1 × 10⁶ cells in 100-mm dishes in 10% FBS-RPMI 1640 and grown overnight. Cells were treated with 20 μM curcumin for 72 h, followed by apoptosis assay using the Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences). Cells treated with vehicle (ethanol) were used as a control. (a) shows a representative FACS assay result for Rh1 cells. (b) Results are means ± SD and are pooled from three independent experiments. *P < 0.05 (unpaired Student's t-test).

Curcumin inhibits the basal and IGF-I-induced motility of rhabdomyosarcoma cells

The most devastating effect of cancer cells arises from their ability to move, leading to cancer cell invasion of local tissues and eventually metastasis to distant sites.56 Curcumin has been shown to inhibit the motility of prostate cancer cells.57 We therefore determined the effect of curcumin on the motility of rhabdomyosarcoma cells. The cell motility was determined by the wound healing assay.51 As shown in Figure 4, Rh1 cells were pretreated with or without 20 μM curcumin for 2 h and then stimulated with or without IGF-I (10 ng/ml) for 22 h. Within 24 h of treatment, curcumin decreased the basal cell motility by ˜70%. IGF-I treatment stimulated Rh1 cell motility by ˜2.7 fold, but curcumin treatment negated this stimulation of motility, bringing Rh1 cellular movement to levels below those of normal basal movement. Taken together, our findings and the data from others suggest that curcumin could act as a powerful anti-metastatic agent against rhabdomyosarcoma and possibly other cancer types.

Figure 4. Curcumin inhibits basal and IGF-I-induced motility of rhabdomyosarcoma cells. A monolayer of Rh1 cells were grown in 6-well plates to 80% confluence, and serum-starved in DMEM for 24 h. Cell motility was assessed by the wound healing assay, as described in Material and methods. (a) Typical photos show that cells migrated over the denuded area under different conditions, as indicated. Bar = 100 μm. (b) Results are means ± SD and are pooled from three independent experiments. *P < 0.05 (unpaired Student's t-test).

Curcumin inhibits the IGF-I stimulated phosphorylation of S6K1, 4E-BP1 and mTOR

Increasing evidence has implicated mTOR as a central controller of cell proliferation, growth, survival and motility.20 The present study and other findings1 have demonstrated that curcumin potently inhibits cell proliferation/growth, survival and motility of rhabdomyosarcoma cells and many other cell lines. In particular, among the proteins regulated by mTOR, a number of them, such as cyclin D1, ODC, c-myc, NF-κB, Akt and PKC, are also targeted by curcumin. We therefore hypothesized that curcumin might be disrupting these cellular processes by primarily inhibiting the mTOR signaling pathway. To test this hypothesis, we set out to examine the effect of curcumin on the mTOR signaling pathway in Rh1 and Rh30 cells. Dose-response and time-course experiments were carried out, followed by Western blot analysis to investigate the effect of curcumin on the individual members of the mTOR signaling pathway. Our results indicate that curcumin inhibited IGF-I-stimulated phosphorylation of S6K1 and 4E-BP1, the two best characterized downstream effector molecules of mTOR, in a dose-dependent manner, starting at 200 nM in Rh1 and Rh30 cells (data not shown). At 2.5 μM, curcumin significantly blocked the IGF-I-stimulated phosphorylation of S6K1 (Thr389, a site regulated by mTOR23, 24) after 2 h exposure (Fig. 5a) and 20 μM curcumin inhibited this phosphorylation event completely within 1 h in Rh30 cells (Fig. 5c). No significant effect of curcumin on total protein levels of S6K1 was detected using an anti-S6K1 antibody that recognizes both phosphorylated and unphosphorylated forms (Fig. 5a, c). Similarly, the effect of curcumin on phosphorylation state of 4E-BP1 was detected with an antibody to 4E-BP1. Phosphorylation of 4E-BP1 decreases its electrophoretic mobility during SDS-polyacrylamide gel electrophoresis.21 Curcumin inhibited IGF-I-stimulated phosphorylation of 4E-BP1 in Rh30 cells, as indicated by the decrease in the intensity of the uppermost band γ and by the increase in the higher mobility band α that corresponds to a less phosphorylated form of 4E-BP1 (Fig. 5a). To confirm this is due to inhibition of mTOR, antibodies to phospho-4E-BP1 at Thr37, Thr46, Ser65 and Thr70, which have been identified as the sites regulated by mTOR,22 were used (Fig. 5b). Our Western blot analysis indicates that curcumin also potently inhibited the phosphorylation of 4E-BP1 at Thr37, Thr46, Ser65 and Thr70 in a dose-dependent manner (Fig. 5b). Taken together, curcumin potently inhibits mTOR-mediated phosphorylation of S6K1 and 4E-BP1.

Figure 5. Curcumin inhibits phosphorylation of S6K1, 4E-BP1 and mTOR more potently than that of Akt. (a) Serum-starved Rh30 cells were treated with curcumin (0–40 μM) for 2 h, followed by stimulation with IGF-1 (10 ng/ml) for 1 h. Cellular extracts were subjected to Western blot analysis with antibodies against p-S6K1 (T389), S6K1, 4E-BP1, β-tubulin (loading control), p-mTOR (S2448), p-mTOR (S2481), mTOR, p-Akt (T308), p-Akt (S473) and Akt, respectively; and (b) with antibodies to 4E-BP1, p-4E-BP1 (T37/46), p-4E-BP1 (S65), p-4E-BP1 (T70) and β-tubulin (loading control), respectively. (c) Rh30 cells grown in 10%FBS-RPMI were treated with curcumin (20 μM) for the indicated time, followed by Western blot analysis with antibodies against to p-S6K1 (T389), S6K1, 4E-BP1 and β-tubulin (loading control), respectively. Left panels show representative blots, while right panels show semiquantitated values of 3–4 independent experiments, by densitometry using NIH ImageJ.

Ser 2448 of mTOR is phosphorylated by Akt,58, 59 whereas Ser 2481 of mTOR is an autophosphorylation site.60 Phosphorylation of mTOR at these two sites has been suggested to be associated with mTOR activity.58, 59, 60 We therefore further examined whether curcumin influences the phosphorylation of mTOR. As shown in Figure 5a, curcumin did dose-dependently inhibit the IGF-I-stimulated phosphorylation of mTOR at Ser2448, and at Ser2481, as detected by Western blot analysis using specific phospho-mTOR antibodies (Ser2448 and Ser2481), respectively.

Consistently, we also found that the IGF-I-stimulated phosphorylation of Akt at Thr308 (phosphorylated by PDK1)61 and Ser473 (phosphorylated by PDK262 or mTOR46) was also inhibited by curcumin, but only at a concentration of 40 μM (Fig. 5a and Fig. 6). The inhibition of Akt phosphorylation by curcumin has been reported by other groups,7, 15 but only at high micromolar concentrations of the compound (50–75 μM). Our results are in good agreement with those findings.

Figure 6. Curcumin inhibits phosphorylation of S6K1, 4E-BP1 and Akt in prostate, breast and cervical cancer cells. (a) Serum-starved DU145 (prostate) and MCF-7 (breast) cells were treated with curcumin (0–40 μM) for 2 h, followed by stimulation with IGF-1 (10 ng/ml) for 1 h. Cellular extracts were subjected to Western blot analysis with antibodies to p-S6K1 (T389), S6K1, 4E-BP1, β-tubulin (loading control), p-Akt (S473) and Akt, respectively. (b) Serum-starved Hela (cervical) cells were exposed to curcumin or rapamycin at indicated concentrations, and then stimulated with IGF-I (10 ng/ml) for 1 h, followed by Western blot analysis with antibodies to p-S6K1 (T389), S6K1, 4E-BP1 and β-tubulin (loading control), respectively.

To exclude the possibility that curcumin inhibition of mTOR signaling is cell-type dependent, prostate (DU145), breast (MCF-7) and cervical (Hela) cancer cells were treated with curcumin (0–40 μM) for 2 h, followed by stimulation with IGF-I (10 ng/ml) for 1 h, respectively. As shown in Figure 6, treatment of cells with curcumin also inhibited IGF-I-stimulated phosphorylation of S6K1 and 4E-BP1 in these cells. Furthermore, curcumin at the concentration of 2.5 μM was as potent as rapamycin at 100 ng/ml to inhibit phosphorylation of S6K1 or 4E-BP1 in Hela cells (Fig. 6b). Similarly, curcumin failed to inhibit IGF-I-stimulated phosphorylation of Akt in DU145 and MCF-7 cells until the concentration reached 40 μM (Fig. 6a). We therefore conclude that curcumin has found a novel cellular target in the downstream components of the mTOR signaling pathway and that this inhibition is occurring at concentrations that are physiologically and clinically relevant. The hitting of this target may explain curcumin's anti-proliferative, pro-apoptotic and anti-motogenic effects on the cancer cells.

Discussion

We have observed that curcumin effectively inhibits rhabdomyosarcoma cell proliferation by arresting the cells in the G₁/G₀ phase of the cell cycle, through a p53-independent mechanism. We have also shown that curcumin induces apoptosis of rhabdomyosarcoma cells with loss of p53 function. This suggests that curcumin may have potential applications as a chemotherapeutic agent against those p53 mutant tumor cells, which are resistant to irradiation therapy or other chemotherapies. We also found that curcumin inhibits that IGF-I stimulated motility of rhabdomyosarcoma cells. This suggests that curcumin is also a potential anti-metastatic agent.

So far, it remains unclear how curcumin inhibits cell proliferation, growth, survival and motility. Studies have revealed numerous cellular proteins targeted by curcumin.1, 2, 3, 4, 6, 7, 8, 9, 11, 12, 17, 18, 19 Of particular interest is that among the proteins regulated by mTOR, a number of them, such as cyclin D1, ODC, c-myc, NF-κB, Akt and PKC, are also targeted by curcumin. Here we show that curcumin inhibits cell growth, induces apoptosis and suppresses motility of rhabdomyosarcoma cells, and concurrently inhibits the signaling pathways (S6K1 and 4E-BP1) mediated by mTOR, a central controller of cell proliferation, growth, survival and motility. The findings suggest that curcumin may execute its anticancer activity by primarily targeting mTOR signaling pathways.

Phase I clinical trials have shown that high doses (8 g/day) of curcumin can be delivered to patients, yielding 1.77 ± 1.87 μM average peak serum concentration, with virtually no deleterious side effects.54 We observed complete abolition of S6K1 and 4E-BP1 phosphorylation and reduced levels of mTOR phosphorylation/autophosphorylation upon 2 h treatment with 2.5 μM curcumin. Our results suggest that the lowest concentration of curcumin that could possibly affect these critical phosphorylation events is well within the range of physiologically achievable concentrations of the compound in cancer patients. We observed curcumin inhibition of Akt phosphorylation only at a concentration of 40 μM, suggesting that at physiological concentrations, curcumin affects these downstream signaling events (and the processes regulated by them) by direct action on these downstream components, but not on inhibition of upstream components (IGF-I receptor, phosphatidylinositol 3′ kinase, Akt) of the mTOR pathway. This represents a novel target for curcumin that could possibly be used for therapeutic benefit in the treatment of rhabdomyosarcoma and other cancers. Our findings indicate that curcumin may represent a new class of mTOR inhibitor. Clearly, more studies are required to elucidate how curcumin inhibits mTOR signaling.

Acknowledgements

This work was supported in part by a Feist-Weiller Cancer Research Award (S.H.), an Edward P. Stiles Award (S.H.) and a Start-up Fund (S.H.) jointly from Louisiana State University Health Sciences Center in Shreveport, LA.